Obtaining precise images of a sample or a sample section, respectively, by means of imaging optics requires the sample to be placed exactly in the focal position of the objective. If the image is out of focus, it is important to find out by what amount and in which direction a change in the position of the sample relative to the imaging optics or relative to the objective, respectively, has to be effected, and, if necessary, to derive corresponding adjusting commands that can be used for re-focusing. In this connection, basically the triangulation method, imaging methods using contrast evaluation, and position determination by means of an inclined confocal slit diaphragm are known. In triangulation methods, a collimated laser beam is reflected into the pupil plane of an objective, and the z-position of the laser light reflected by the sample is deduced from the course of said laser beam relative to the imaging beam path. However, during imaging of the laser light into planes of the sample located at different depths, image defects occur so that the autofocus quality varies significantly over a given depth-of-focus range. Also, variations can be established as to whether the result of measurement is determined from the middle or on the edge of the sample or of the detector used, respectively. Therefore, a triangulation method is usually carried out iteratively, which is relatively time-consuming.
In imaging methods with contrast evaluation, the sample is illuminated with a specific intensity distribution, generally by placing a grating in a field aperture plane of an illumination beam path. A series of images is recorded with different distances between the imaging optics and the sample and, among the images in this series, the one having the highest contrast is determined to which the optimal focal distance is then assigned. This has the disadvantage of requiring high-precision placement at different z positions so as to record the series of images, which is in turn time-consuming. Examples of an autofocus device analyzing the contrast of a pattern projected onto a sample are to be found in U.S. Pat. Nos. 5,604,344 or 6,545,756.
During position determination by means of an inclined confocal slit diaphragm, a slit diaphragm is placed in a field aperture plane of the illumination beam path and is imaged onto the sample. The light reflected by the sample is directed onto a CCD line that is inclined relative to the slit diaphragm and the position on the CCD line is determined at which the reflected light has a maximum. This method is very quick; however, it does have problems with impurities on the sample or sample surface that may lead to intensity variations. Also, a very large amount of adjustment is required when imaging the slit onto the CCD line, because the slit has to be very narrow in order to be able to achieve high precision. An improvement to position determination by means of an inclined confocal slit diaphragm is described in DE 10319182 A1.
All these methods have in common that, while they can find the focal plane very precisely, they allow determination of said focal plane within the sample, especially with respect to further interfaces, only to a limited extent.
However, in many cases it is desired not only to exactly find the plane of measurement, but also to determine the position of said plane, i.e. its distance from a reference plane. In the prior art, referencing to an interface which serves as a reference plane can be effected by using a second autofocus device being focused on the interface. This naturally increases optical complexity and generally makes it necessary to reserve an area of the detection or illumination aperture, respectively, for this additional autofocus. The use of several autofocus beam paths is described, for example, in WO 00/43820. On the other hand, it is known in the state of the art to interrupt measurement for a short time and to adjust the autofocus device to the desired reference plane by a focus adjustment. The absolute value of the focus adjustment then represents a measure for the distance from the measurement plane to the reference plane. This has the disadvantage that the actual microscopic measurement for determining the distance from the reference plane has to be interrupted.
The same problems arise when an object that may be present at different z-positions has to be found or traced in a sample. Such an object may be, for example, a cell present in a sample and moving in the sample (e. g. in a liquid solution).